Fig 1.
Barrier properties of H441 and A549 cells.
(A) TEER and (B) TEPD across H441 cell and A549 cell monolayers measured in the culture period of 0–15 days (n = 9). (C) Time course (n = 6) and (D) apparent permeability (n = 11) of sodium fluorescein across H441 and A549 monolayers measured in the apical-to-basolateral direction on Day 9. Data are shown as mean ± SD; ****, P < 0.0001 by Student’s t- test.
Fig 2.
mRNA expression profiles of NCI-H441 and A549 cells.
mRNA samples (n = 3) were obtained from three independent cultures of H441 cells and A549 cells. mRNA expression of (A) alveolar epithelial cell markers, (B) junctional proteins, (C) Na+ channels and (D) Cl- channels were analysed by quantitative real-time PCR. Data are shown as mean ± SD; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA analysis followed by post-hoc Bonferroni test. (#: target not detected.)
Fig 3.
Protein expression profiles of NCI-H441 and A549 cells.
Expression of (A) junctional proteins, (B) Na+ and water transporters and (C) CFTR was examined by Western blotting. Protein lysates from three independent cultures of H441 cells (Lane 1, 2 and 3) and A549 cells (Lane 4, 5 and 6) are tested (n = 3). In (C) Calu-3 cell lysates were used as positive control (Lane 7). Levels of actin expression were used to monitor protein loading. The size of the closest molecular weight marker to each target protein is shown.
Fig 4.
Immunostaining of H441 and A549 cells for ZO-1, E-cadherin and α1-Na+-K+-ATPase.
(A, B) H441 cells and A549 cells were double stained for either ZO-1 and α1-Na+-K+-ATPase or (C, D) ZO-1 and E-cadherin. (a) The locations of the XY slices are shown in the schematic YZ section. Nuclei are stained blue. (b, c) XY and (d–f) YZ scans are shown. Representative images from two independent experiments were shown.
Fig 5.
Optimisation of cell culture conditions.
(A) Effect of seeding density on the development of H441 cell layers. Nuclei are stained blue. Cross-sections from YZ scans are shown. Effect of ITS on (B) TEER (n = 5), (C) TEPD (n = 5) and (D) protein expression in the presence and absence of dexamethasone. (E) Dose response of TEER (n = 7), (F) TEPD (n = 7) and (G) protein expression to dexamethasone in the presence of ITS. Protein expression data show representative images from two independent experiments. Data are shown as mean ± SD; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 using one-way ANOVA analysis followed by post-hoc Dunnett test.
Fig 6.
Na+ and Cl− transport under baseline and stimulated conditions across H441 cells.
(A) Response of TEPD to different perturbations. Each agent was used at a concentration of 10 μM (n = 13 for amiloride; n = 6 for ouabain, CFTR (inh)-172 and NPPB). Dose response of TEPD to: (B) ENaC inhibitor amiloride (n = 5), (C) CFTR inhibitor CFTR (inh)-172 (n = 6) and (D) broad spectrum chloride channel inhibitor NPPB (n = 6). (E) Effect of forskolin followed by amiloride, CFTR(inh)-172 or NPPB on TEPD (n = 9). (F) Effect of amiloride followed by forskolin, CFTR(inh)-172 or NPPB on TEPD (n = 7). (CTL: control; A: amiloride; O: ouabain; C: CFTR (inh)-172; N: NPPB; F: forskolin). Results are combined from three independent experiments. Data are shown as mean ± SD; **, P < 0.01; ****, P < 0.0001 using one-way ANOVA analysis followed by post-hoc Dunnett test.